This invention relates to an apparatus and method for isolating and recovering an objective cell intended to obtain from a liquid to be treated.
When a cell is treated in vitro as in the case of a cell-using therapy, for example, hematopoietic stem cell transplantation, adoptive immunotherapy, gene therapy, etc., or in the case of the culture and preservation of a cell, the isolation of an objective cell intended to be recovered (hereinafter referred to as an objective cell) such as lymphocyte from other components and subsequent concentration of the objective cell have been usually performed.
In the culture of a cell or in the cell-using therapy, it has been a very important subject matter to remove cells other than an objective cell, and further, to remove unwanted liquid components as well as the wastes and metabolic products of cells. In the employment of a preserved cell, it has been also a very important subject matter to remove materials harmful to cells or living body. An example of such harmful material is a cryoprotective agent which is employed for cryopreservation. In view of these problems, there have been proposed various methods of cell-isolation and recovery methods corresponding to these cell-isolation.
The method for isolating cell can be roughly classified into (1) a method based on differences in specific density of cells, such as a sedimentation method, a centrifugation method and a density gradient centrifugation method; (2) an electric separation method based on the surface electric charge of cells; (3) an affinity separation method taking advantage of the antibody specific to the surface antigen of the cells; and (4) a filtering separation method based on the differences in size and in deformability of cells.
These methods (1) to (4), however, are accompanied with the following defects.
In the case of the sedimentation method (1) which is based on the differences in the density of cells, since the objective cell is to be separated by gravity, it takes a long period of time for the isolation of the objective cell, and the isolation efficiency is very poor. Additionally, there are problems that the purity and yield of the objective cell are also low.
With a view to improve the isolation efficiency by using centrifugal force, there has been developed the centrifugation method, which is generally employed as a method for treating a large quantity of cells. However, since this centrifugation method requires a large-scaled and expensive apparatus for an aseptic treatment and recovery of cells, and since differences in density among cells are not so large, the types of cells that can be separated are inevitably restricted.
When it is desired to improve the separability, use is made of the density gradient centrifugation method where a specific density medium whose specific density is strictly adjusted. However, this method is accompanied with problems that it cannot simultaneously treat a large quantity of cells and involves a very complicated operation. Specifically, the objective cell is required to be carefully recovered from an interface formed by density-differences; the operation of removing unwanted cell components is required to be performed in a separate condition from that of removing unwanted liquid components; and the sterility in the recovery operation cannot be ensured unless a clean bench is employed.
Further, in the aforementioned method (1), depending on the condition of the centrifugation, the objective cell may be badly damaged.
In the case of electric separation method (2), since the differences in surface electric charge among the cells are not so large, the separability of the cells is inevitably restricted. Further, the method is not suited for promptly treating a large quantity of cells. Additionally, there is a probability in this method that the objective cell may be badly damaged due to the application of an electric field to the objective cell.
The affinity separation method (3) is most excellent in view of specificity among the aforementioned various isolation methods. However, if the isolated cell is to be recovered using this method, an enzymatic treatment for cleavage an antibody molecule combined to the cell is required. Therefore, it will be confronted with technical problems such as the generation of damage to the objective cell due to the enzymatic treatment, a troublesome operation and the maintenance of the activity of antibody. Additionally, since it employs an expensive antibody, the cost for the isolation of cell will be increased, thus making the method inappropriate for a prompt treatment of a large amount of cells.
The filtering separation method (4) is featured in that it comprises the steps of passing a suspension liquid containing an objective cell through a filtering member so as to capture the objective cell on the filtering member, passing recovering liquid through the filtering member in the direction opposite to that of the capturing step so as to remove the objective cell captured on the filtering member, and recovering the objective cell. Although this filtering separation method is suited for promptly separating a large quantity of objective cell from the unwanted cells as well as from unwanted liquid components, this method is defective in that the recovery ratio of the objective cell is low. This defect can be ascribed to the fact that the fine pore diameter of the filtering member is constant throughout a sequence of the treatment procedures.
Specifically, when the pore size of the filtering member is set relatively larger with a view to improve the releasability of the objective cell at the step of recovering, the amount of the objective cell that passes through the filtering member without being captured by the filtering member will be increased. On the contrary, if the pore size of the filtering member is set relatively smaller, the amount of the objective cell that passes through the filtering member will be minimized, thereby increasing the quantity of objective cell that can be captured by the filtering member. However, the adhesivity of the objective cell to the fine pore of the filtering member will be increased so that the releasability of the objective cell at the step of recovering the objective cell will be deteriorated, thus making it impossible to recover the objective cell at a high yield. In addition, the removability of unwanted cells will be also deteriorated.
Under the circumstances, depending on the type of the objective cell, a filtering member having a suitable pore size which is capable of satisfying not only the capturability but also the releasability of the objective cell is required to be selected.
Incidentally, it is possible in this filtering separation method to decrease more or less the quantity of the objective cell that passes through the filtering member and to increase the capturing ratio of the objective cell by reducing the quantity of feeding the suspension of cells on the occasion of the filtration. However, if the feeding quantity of the suspension of cells is reduced, the throughput per unit time of the suspension of cells is also caused to decrease, thus spoiling the advantage of the filtering separation method, i.e. a prompt treatment of large quantity of the suspension of cells.
It is also possible in this filtering separation method to improve the releasability of the objective cell that has been captured on the filtering member by increasing the feeding quantity of the recovery liquid on the occasion of the recovery of the objective cell. However, if the feeding quantity of the recovery liquid is increased, the objective cell may be increasingly damaged, thus deteriorating the characteristics and quality of the recovered objective cell.
As described above, the prior arts have the merits and demerits in the means for isolating the objective cell from the unwanted components as well as in the means for recovering the isolated cell. Under the circumstances, the aforementioned methods have been suitably selected or suitably combined depending on the purpose and the required level of isolation.